Many human diseases, such as developmental disorders, cancer and neurological diseases, are caused by genetic mosaicism, in which the disease-causing cells carry distinct gene mutations from the rest of the body. Therefore, genetic mosaic animal models are highly valuable for both basic and translational research. For basic research, genetic mosaic models reveal in vivo behaviors of mutant cells to facilitate our understanding of both gene functions in normal biological processes and genetic mechanisms of disease etiology. For translational research, genetic mosaic models can be used for preclinical testing of therapeutic strategies for their efficacy in preventing, treating, and even reversing diseases. While Cre-loxP based conditional knockout models as genetic mosaics have led to many groundbreaking discoveries, the resolution is often at the tissue- level, which creates significantly challenges for cellular resolution phenotypic analysis. To overcome this problem, previously we have established a genetic mosaic system in mouse, termed Mosaic Analysis with Double Markers (MADM, Zong 2005 Cell). From a colorless, heterozygous mouse, the MADM system generates sparse GFP+ mutant and RFP+ WT sibling cells through inter-chromosomal mitotic recombination. Sparse labeling and 100% color-genotype matching enable in vivo phenotypic analysis at the single-cell resolution. MADM was broadly adopted in many fields such as neurobiology (Hippenmeyer 2010 Neuron), developmental biology (Packard 2013 Developmental cell), and cancer biology (Liu 2011 Cell). While we have learned a lot of fascinating biology with the mouse MADM system, zebrafish as a model organism carries great advantages in terms of the transparency of its body and the ease to generate a large, genetically identical population. If one could establish a zebrafish equivalent of the mouse MADM system, live imaging could be readily used to gain dynamic information of in vivo gene functions and disease-progression mechanisms. As importantly, high-throughput screening could be readily set up to identify drug candidates that could stop or even reverse the disease progression in such a model with a multi-well, high-content imaging based platform. To establish the zebrafish MADM system, in Specific Aim 1, we will construct the MADM cassettes tailored for zebrafish and precisely target two MADM alleles at the pre-selected identical locus in the genome with the CRISPR-CAS9 system to establish the zebrafish MADM system; and in Specific Aim 2, we will perform basic characterization of the zebrafish MADM system after breeding a few Cre transgenes into the founder zebrafish, including the level of reporter gene expression, recombination efficiency in various tissues, etc. Upon the establishment of the zebrafish MADM, we will deposit it into public repository such as ZIRC for all labs in the field to use. We envision that the system will have a significant impact on fields including developmental biology, neuroscience, cancer biology, regenerative medicine, and much more.